Following infection with most reovirus strains, viral protein synthesis is robust, even when cellular translation is inhibited. To gain further insight into pathways that regulate translation in reovirus-infected cells, we performed a comparative microarray analysis of cellular gene expression following infection with two strains of reovirus that inhibit host translation (clone 8 and clone 87) and one strain that does not (Dearing). Infection with clone 8 and clone 87 significantly increased the expression of cellular genes characteristic of stress responses, including the integrated stress response. Infection with these same strains decreased transcript and protein levels of P58 IPK , the cellular inhibitor of the eukaryotic initiation factor 2␣ (eIF2␣) kinases PKR and PERK. Since infection with host shutoff-inducing strains of reovirus impacted cellular pathways that control eIF2␣ phosphorylation and unphosphorylated eIF2␣ is required for translation initiation, we examined reovirus replication in a variety of cell lines with mutations that impact eIF2␣ phosphorylation. Our results revealed that reovirus replication is more efficient in the presence of eIF2␣ kinases and phosphorylatable eIF2␣. When eIF2␣ is phosphorylated, it promotes the synthesis of ATF4, a transcription factor that controls cellular recovery from stress. We found that the presence of this transcription factor increased reovirus yields 10-to 100-fold. eIF2␣ phosphorylation also led to the formation of stress granules in reovirus-infected cells. Based on these results, we hypothesize that eIF2␣ phosphorylation facilitates reovirus replication in two ways-first, by inducing ATF4 synthesis, and second, by creating an environment that places abundant reovirus transcripts at a competitive advantage for limited translational components.As representative members of the Reoviridae family, mammalian orthoreoviruses (reoviruses) have genomes composed of 10 segments of double-stranded RNA (dsRNA) that are surrounded by two concentric protein capsids (reviewed in reference 54). In natural infections, reoviruses replicate in cells of the respiratory or enteric tract; however, in experimental infections of neonatal mice, their tropism is much broader. Pathogenesis studies with the mouse model have demonstrated that reoviruses replicate in cells of the brain, heart, liver, muscle, and pancreas (reviewed in reference 73). At the cellular level, the consequences of reovirus infection have been extensively analyzed and include the inhibition of DNA synthesis, cell cycle arrest at the G 2 /M stage, apoptosis induction, translational inhibition, and type